The pepsin enzyme is found in the stomach, with the aid of hydrochloric acid, which provides a low pH environment for pepsin to operate in. The stomach performs digestion typically at body temperature, which is about 37 degrees. Therefore, by recreating conditions similar to the stomach, I expect to find the pepsin enzyme will be most efficient at 40 degrees, the temperature which the experiment is carried out that is closest to body temperature. As the temperature drops the pepsin enzyme will have less kinetic energy therefore slowing down the reaction, increasing the time taken, but if the temperature increases it will function fine again, as no bonds will have been broken. If the temperature increases above 40 degrees the reaction time will also increase again, and if it does so dramatically then the enzyme will become denatured.
Apparatus:
5cm cubed egg albumen
5cm cubed pepsin 5%
2 drops of: HCl 2M
Test tubes
Pipette
Stopwatch
Universal Indicator Paper
Measuring cylinder
Water baths heated to: 20 degrees
30 degrees
40 degrees
50 degrees
60 degrees
Diagram:
Method:
Measure out 5cm squared of egg albumen in a measuring cylinder, and then 5cm squared of pepsin 5% separately. Heat them both individually for 2 to 3 minutes at the desired temperature waterbaths, which should be electronic so as to keep consistent temperature. For 20 degrees room temperature can be used, i.e. no waterbath; and for 30 degrees water from an above temperature will need to be cooled with ice (or colder water heated) and then checked constantly so that it stays at 30 degrees. Now add these two together in a single tube. To this add 2 drops of hydrochloric acid, but before so, test a drop of the hydrochloric acid on some universal indicator paper to check that it is pH2. This experiment must be repeated with 5 changes of temperature:
- 20 degrees
- 30 degrees
- 40 degrees
- 50 degrees
- 60 degrees
Each variation should be repeated 3 times to check for consistency, from these 3 results of each change find an average.
At first the mixture should be cloudy white due to the whiteness of the egg albumen, start timing when the mixture is first composed and the reaction/timing is finished when the mixture goes clear. This is as all the protein in the egg albumen has been digested into amino acids (which are colourless) due to the pepsin.
Fair Test:
So that the experiment is fair, the same amount of all solutions will be used in each experiment. All experiments will also be carried out at the same pH (2M hydrochloric acid – pH 2) as if this is changed then this may cause the denaturing of the enzyme or slow the reaction giving anomalous results. The input variable will be the temperature, and the output variable the time taken for digestion. Each temperature level will also be repeated 3 times, to check for consistency in the results
Results:
Conclusion:
My graph shows a smooth curve, that does not fit my prediction that 40 degrees would be the optimum temperature, and instead suggests that 60 degrees is the optimum temperature, or perhaps even higher (no temperatures above 60 were used); but it does still show the rate of reaction increases with temperature to the optimum temperature, just that this time the optimum temperature is higher. And as the optimum temperature is higher, this means
This shows therefore that the enzyme did not become denatured at 50 degrees, because it could not have performed so successfully at such a high temperature otherwise. Since other scientists have proved that the optimum temperature for pepsin to perform at is 40 degrees in the human body, the only plausible explanation is that this pepsin was not from a human. Instead, I believe that this pepsin came from another animal with a higher body temperature, about 60 degrees or higher, as this would explain the otherwise anomalous readings.
Nevertheless, the pepsin did still perform its function as the egg albumen turned clear, albeit at a higher optimum temperatures, the only difference is its origin, its function is the same. This indicates the egg albumen had been converted to its simpler form, amino acids:
Evaluation:
Although my results do not support my prediction, I believe that my methodology used to obtain them was correct and was efficient. The only thing I would change in my experimental set up is the type of pepsin used, meaning that I would change from an unspecified origin pepsin to a humans digestive pepsin, as this is what my predictions had been based upon, therefore meaning that I was wrong before I had even started. This is as the type of pepsin (its origin) has a determining effect on its optimum temperature, pepsin from the human body will have an optimum temperature of 38 degrees, but for example, a pigs may be different, and again different to that of pepsin ‘grown’ from bacteria in a lab. Although this could have been avoided if I had checked upon the origin of the pepsin.
In addition to this, I would like to experiment with this type of pepsin at temperatures more varied, i.e. ranging far above 60 degrees so that I could find out at which point it denatures, and also to do more frequent tests around 60 degrees, so as to find out the optimum temperature.
From my experiment, I believe that really I produced a set of results, that, compared against the prediction for my experiment are anomalous after the result for 40 degrees, but as the pepsin used was not what my prediction had its origins in, I believe that really none of the results therefore can be considered wrong as the results did still follow the trends for changes in temperatures on enzymes.
If it had have been possible, I would also had liked to use electronic water baths for all temperatures, as that this would have insured constant temperatures, whereas as this was not so for several temperatures, constant monitoring of a thermometer had to be undertaken, so that if the temperature suddenly changed, methods could be taken to rectify this; but for the seconds when it was a degree or two above/below the required it was an unfair test.
Also, if I was to repeat the experiment then I would have used precise measurements of hydrochloric acid, rather than imprecise measurements such as two drops, as the size of a drop depends on several factors, such as the amount of acid in the pipette as if there is little then the drops will be less etc and also the size of pipette or dropper used as obviously the larger the hole the more acid will fall through. A method more suitable would have been to measure a set amount of acid into a measuring cylinder, then each time allow a set equal amount into the pipette and ensure all of this acid is added each time.
